Cd133 related to anticancer agent resistance in colon cancer and use thereof

ABSTRACT

The present invention relates to a use of CD133 involved in resistance to an EGFR-targeting agent in colon cancer. The CD133 protein may be used as a novel target protein for diagnosing and treating resistant cancer as well as general cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Rule 53(b) Divisional of U.S. application Ser. No.16/848,273 filed Apr. 14, 2020, which claims priority based on KoreanPatent Application No. 10-2019-0043765 filed Apr. 15, 2019. The contentsof all of the above-identified applications are incorporated herein byreference in their entireties.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The content of the electronically submitted sequence listing, file name:Q285877_sequence listing as filed.XML; size: 7,331 bytes; and date ofcreation: Mar. 17, 2023, is hereby incorporated by reference in theirentirety.

TECHNICAL FIELD

The present invention relates to a use of CD133 involved in anticanceragent resistance in colon cancer.

BACKGROUND ART

Although many anticancer agents against various types of cancer havebeen developed to date, only a few types of cancer can be completelycured with an anticancer agent alone, this is because cancer cells donot respond to an anticancer agent in cancer treatment with theanticancer agent, or tumors are effectively reduced in an early stage,but develop anticancer agent resistance during or after treatment.Therefore, for effective anticancer treatment, resistance to ananticancer agent, for example, anticancer agent resistance of cancercells, has to be overcome.

Generally, a mutation in the KRAS, NRAS or BRAF gene results inproduction of a protein having a modified signaling characteristic intumor cells, and such a mutation has been known to be associated withunsuccessful results in cancer treatment using a therapeutic antibodytargeting an epithelial growth factor receptor (EGFR), for example,gefitinib, cetuximab or panitumumab (Amado, Wolf et al. 2008; Karapetis,Khambata-Ford et al. 2008; Di Nicolantonio, Martini et al. 2008;Loupakis, Ruzzo et al. 2009; Lievre, Bachet et al. 2006). For example,KRAS G13D known as a colon cancer mutant cell line was reported to haveresistance (tolerance) to an anticancer agent targeting EGFR.

For this reason, to improve a therapeutic effect of an anticancer agenttargeting EGFR for cancer treatment, it is urgent to solve such aproblem of anticancer agent resistance. Research on the identificationof a specific cause or mechanism of the anticancer agent resistance hasbeen persistently conducted, but it is a reality that little is knownabout it.

Nevertheless, the inventors had identified that, through a resistancestudy for colon cancer mutant cell lines resistant to an anticanceragent targeting EGFR, CD133 is related to resistance of colon cancercells against an anticancer agent targeting EGFR, and thus the presentinvention was completed.

TECHNICAL PROBLEM

The present invention is directed to providing a biomarker compositionfor diagnosing resistance to an EGFR-targeting agent in colon cancer,which includes CD133 protein or a gene encoding the same.

The present invention is also directed to providing a kit for diagnosingresistance to an EGFR-targeting agent in colon cancer.

The present invention is also directed to providing a method ofproviding information required for diagnosis of resistance to anEGFR-targeting agent in colon cancer.

TECHNICAL SOLUTION

The inventors had confirmed, through a study on a KRAS G13D mutant coloncancer cell line having resistance to an EGFR-targeting agent, that thecell migration to and invasiveness of adjacent normal cells are highlyimproved by transferring the mutant to adjacent cells via microvesicles,the migration of KRAS G13D via microvesicles imparts anticancer agentresistance to adjacent colon cancer cells, and the CD133 proteincontrolling the release of microvesicles is related to resistance to anEGFR-targeting agent, and thus the present invention was completed.

The present invention is characterized by confirming whether coloncancer cells have resistance to an EGFR-targeting agent using theactivity of the CD133 gene and an active protein, which is a productthereof, as a biomarker.

To this end, the present invention provides a biomarker composition fordiagnosing resistance to an EGFR-targeting agent in colon cancer, whichincludes the CD133 protein or a gene encoding the same.

The biomarker of the present invention may be a marker with anticanceragent resistance, which is an EGFR-targeting agent, and since it hasexcellent accuracy and reliability as a marker for diagnosing anticanceragent resistance, the biomarker may be used to diagnose the occurrence,development and/or metastasis of resistant cancer, and treat resistantcancer.

The term “resistance” or “tolerance” used herein means that an organismdoes not sensitively respond to a drug, and thus withstands the drug'seffects.

CD133 used as a biomarker for resistance diagnosis in the presentinvention is one of the representative CD-type proteins expressed on thesurface of cancer stem cells, which are reported to be mainly present incancer stem cells of colon cancer, liver cancer, pancreatic cancer andlung cancer.

The term “colon cancer” used herein is a collective term referring torectal cancer, colorectal cancer and anal cancer.

In addition, the EGFR-targeting agent of the present invention refers toan anticancer agent, which may be any EGFR-targeting agent exhibiting ananticancer effect, and is used interchangeably with an EGFR-targetingdrug. Preferably, the EGFR-targeting agent is one or more selected fromthe group consisting of cetuximab, gefitinib, erlotinib, panitumumab,PKI-166, EKB-569, HKI-272 (WAY-177820), icotinib, brigatinib, afatinib,lapatinib, canertinib, AEE788, XL647, and Zactima. More preferably, theEGFR-targeting agent is cetuximab, gefitinib, erlotinib or panitumumab,and most preferably, gefitinib.

The term “diagnosis” used herein refers to the detection of apathological condition, which means, in terms of the purpose of thepresent invention, the determination of the presence or absence ofresistance, and the development or alleviation of symptoms of a diseaseby examining the presence or absence of the expression of a biomarkerfor diagnosing resistance to an EGFR-targeting agent.

The “diagnosis biomarker” used herein refers to a material that can beused in diagnosis by distinguishing the presence or absence ofresistance to an EGFR-targeting agent, and includes organic biomoleculessuch as polypeptides or nucleic acids (e.g., mRNA, etc.), lipids,glycolipids, glycoproteins, saccharides (a monosaccharide, adisaccharide, an oligosaccharide, etc.), which increase or decrease incancer cells resistant to an anticancer agent, compared to cancer cells.The biomarker for resistance diagnosis according to the presentinvention may be a protein expressed from the CD133 gene whoseexpression level is increased in resistant cancer cells with respect toan EGFR-targeting agent, compared to general cancer cells.

The composition for diagnosing resistance to an EGFR-targeting agent mayinclude an agent that measures an mRNA expression level of the CD133gene or an amount of a protein expressed from the gene, and such anagent may be an oligonucleotide having a complementary sequence to CD133mRNA, for example, a primer or nucleic acid probe specifically bindingto CD133 mRNA, or an antibody specific for CD133 protein.

The primer refers to a single-stranded oligonucleotide that is able toserve as a starting point of template-directed DNA synthesis undersuitable conditions (that is, four different types of nucleosidetriphosphates and a polymerase) in a suitable buffer at a suitabletemperature. A suitable length of the primer may vary according tovarious factors, for example, a temperature and the usage of the primer.In addition, the primer sequence is not required to be perfectlycomplementary to a partial sequence of the template, and it issufficient that the primer sequence has sufficient complementaritywithin a range in which the primer can do an intrinsic action whenhybridized with the template. Therefore, the primer of the presentinvention does not need to have a perfectly complementary sequence tothe nucleotide sequence of a gene, which is the template, and it isreasonable that the primer has a sufficient complementarity within arange in which the primer can do an intrinsic action when hybridizedwith the gene sequence. In addition, the primer according to the presentinvention is preferably used in a gene amplification reaction. Theamplification reaction refers to a reaction of amplifying a nucleic acidmolecule, and amplification reactions of such a gene are well known inthe art, and may include, for example, polymerase chain reaction (PCR),reverse transcriptase chain reaction (RT-PCR), ligase chain reaction(LCR), transcription-mediated amplification (TMA), and nucleic acidsequence-based amplification (NASBA).

The nucleic acid probe refers to a natural or modified monomer or linearoligomer consisting of linkages of such monomers, and includes adeoxyribonucleotide and a ribonucleotide, may be specifically hybridizedwith a target nucleotide sequence, and naturally occurs or isartificially synthesized. The probe according to the present inventionmay be a single chain, and preferably, an oligodeoxyribonucleotide. Theprobe of the present invention may include natural dNMPs (i.e., dAMP,dGMP, dCMP and dTMP), or nucleotide analogs or derivatives. In addition,the probe of the present invention may include a ribonucleotide. Forexample, the probe of the present invention may includebackbone-modified nucleotides, such as a peptide nucleic acid (PNA),phosphorothioate DNA, phosphodithioate DNA, phosphoroamidate DNA,amide-linked DNA, MMI-linked DNA, 2′-O-methyl RNA, a-DNA and methylphosphonate DNA; sugar-modified nucleotides, such as 2′-O-methyl RNA,2′-fluoro RNA, 2′-amino RNA, 2′-O-alkyl DNA, 2′-O-allyl DNA,2′-O-alkynyl DNA, hexose DNA, pyranosyl RNA and anhydrohexitol DNA;base-modified nucleotides, such as pyrimidines with a C-5 substituent(the substituent may be fluoro-, bromo-, chloro-, iodo-, methyl-,ethyl-, vinyl-, formyl-, ethynyl-, propynyl-, alkynyl-, thiazolyl-,imidazolyl-, or pyridyl-), 7-deazapurines with a C-7 substituent (thesubstituent may be fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-,vinyl-, formyl-, alkynyl-, alkenyl-, thiazolyl-, imidazolyl-, orpyridyl-); inosine; and diaminopurine.

The CD133-specific antibody may be a polyclonal antibody, a monoclonalantibody, a human antibody or a humanized antibody.

Examples of the antibody fragments include Fab, Fab′, F(ab′)₂ and Fvfragments; a diabody; a linear antibody (Zapata et al., ProteinEng.8(10):1057-1062(1995)); a single-chain antibody molecule; and amulti-specific antibody formed from an antibody fragment.

When an antibody is digested with papain, two identical antigen-bindingfragments, that is, “Fab” fragments having a single antigen-bindingregion, and the remainder, “Fc” fragment, are obtained. When treatedwith pepsin, a F(ab′)₂ fragment which has two antigen-binding regionsand is still able to be crosslinked to an antigen is produced. Fv is aminimal antibody fragment including a complete antigen-recognizing andbinding region. This region consists of a dimer of one heavy chainvariable region and one light chain variable region, which are firmlybound to each other by a non-covalent bond.

A method of preparing a polyclonal antibody is known to those ofordinary skill in the art. The polyclonal antibody may be prepared byone or more injections of an immunizing agent in combination with animmune adjuvant, if necessary, into a mammal. Generally, an immunizingagent and/or an immune adjuvant is/are subcutaneously orintraperitoneally injected into a mammal several times. The immunizingagent may be a protein of the present invention or a fusion proteinthereof It may be effective that an immunizing agent, as well as aprotein known to be immunogenic, is injected into an immunized mammal.

The monoclonal antibody according to the present invention may beprepared by the hybridoma method disclosed in the literature (Kohler etal., Nature, 256:495 (1975)), or a recombinant DNA method (refer to U.S.Pat. No. 4,816,576). The monoclonal antibody may also be isolated from aphage antibody library using the technology disclosed in the literature(Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol.Biol., 222:581-597 (1991)).

In the monoclonal antibody of the present invention, specifically, whenexhibiting desired activity, a part of a heavy chain and/or a lightchain is identical to or has homology with a corresponding sequence ofan antibody derived from a specific species or an antibody belonging toa specific antibody class or subclass, whereas the remainder of thechain(s) includes an antibody derived from a different species, anantibody belonging to a different antibody class or subclass or a“chimeric” antibody which is identical or homologous to a fragment ofsuch an antibody (Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)).

A “humanized” form of a non-human (e.g., rodents) antibody is a chimericimmunoglobulin including a minimal sequence derived from a non-humanimmunoglobulin, an immunoglobulin chain or a fragment thereof (e.g., Fv,Fab, Fab′, F(ab′)₂ or a different antigen-binding sequence of anantibody). In most cases, the humanized antibody includes a humanimmunoglobulin (recipient antibody) in which acomplementarity-determining region (CDR) residue of a recipient issubstituted with a CDR residue of a non-human species (donor antibody)such as a mouse, rat or rabbit, having desired specificity, affinity andability. In some cases, aFv framework residue of the humanimmunoglobulin is substituted with a corresponding non-human residue. Inaddition, the humanized antibody may include a recipient antibody, or aresidue which is not found in an introduced CDR or framework sequence.Generally, the humanized antibody substantially includes one or more,generally two or more variable domains, and here, all or substantiallyall CDR regions correspond to a region of a non-human immunoglobulin,and all or substantially all FR regions correspond to a region of ahuman immunoglobulin sequence. In addition, the humanized antibodyincludes at least a part of a variable region (Fc) of an immunoglobulin,generally, a part of a human immunoglobulin region (Presta, Curr. Op.Struct. Biol. 2:593-596 (1992)).

The composition for diagnosing resistance to an EGFR-targeting agentaccording to the present invention may be included in the form of a kit.

The kit may include a primer, probe or antibody which is able to measurethe expression level of the CD133 gene or an amount of the CD133protein, and the definition thereof is as described above.

When the kit is employed in PCR amplification, optionally, reagentsrequired for PCR amplification, for example, a buffer, a DNA polymerase(e.g., a heat-stable DNA polymerase obtained from Thermusaquaticus(Taq), Thermusthermophilus (Tth), Thermusfiliformis, Thermisflavus,Thermococcusliteralis or Pyrococcusfuriosus (Pfu)), a DNA polymerasecofactor and dNTPs may be included, and when the kit is used in animmunoassay, the kit of the present invention may optionally include asecondary antibody and a marker substrate. Further, the kit according tothe present invention may be manufactured in multiple individualpackages or compartments containing the above-mentioned reagentcomponents.

In addition, the composition for diagnosing resistance to anEGFR-targeting agent according to the present invention may be includedin the form of a microarray.

In the microarray of the present invention, a primer, probe or antibodythat is able to measure the expression level of the CD133 protein orgene encoding the same is used as a hybridizable array element, andfixed on a substrate. A preferable substrate may be a suitable rigid orsemi-rigid support, for example, a membrane, a filter, a chip, a slide,a wafer, a fiber, a magnetic or non-magnetic bead, a gel, a tubing, aplate, a polymer, a microparticle and a capillary. The hybridizablearray element may be arranged and immobilized on the substrate, and suchimmobilization may be performed by a chemical binding method or acovalent binding method such as UV. For example, the hybridizable arrayelement may be bound to a glass surface which is modified to include anepoxy compound or an aldehyde group, or bound to a polylysine-coatedsurface using UV. In addition, the hybridizable array element may bindto a substrate by a linker (e.g., an ethylene glycol oligomer or adiamine).

Meanwhile, when a sample applied to the microarray of the presentinvention is a nucleic acid, it may be labeled and hybridized with anarray element fixed on the microarray. Hybridization conditions mayvary, and detection and analysis of a hybridization degree may beperformed in various ways according to a labeling material.

In addition, the present invention provides a method of providinginformation for diagnosing resistance to an EGFR-targeting agent, whichincludes measuring the expression level of the CD133 gene or theexpressed protein in a biological sample isolated from a patient, andmore specifically, the method may include (a) measuring the expressionlevel of the CD133 gene or an amount of the expressed protein in abiological sample of a patient; and (b) measuring the expression levelof the gene and an amount of the expressed protein from a sample of anormal control and comparing it with the measurement result obtained inStep (a).

The method of measuring the expression level of a gene or an amount of aprotein encoded by the gene may be performed by a known process ofisolating mRNA or a protein from a biological sample using a knowntechnique.

The biological sample refers to a sample obtained from the living body,which is different from a normal control in terms of the expressionlevel of the gene or a protein level according to the occurrence orprogression of resistance to an EGFR-targeting agent, and the sample maybe, but is not limited to, tissue, a cell, blood, serum, plasma, salivaor urine.

The measurement of the expression level of a gene is preferably tomeasure an mRNA level, and methods of measuring an mRNA level include,but are not limited to, RT-PCR, real time reverse transcriptionpolymerase chain reaction, RNase protection assay, Northern blotting andDNA chip assay.

The measurement of a protein level may use an antibody, and in thiscase, the CD133 protein in a biological sample and an antibody specificfor the protein forms a binding product, that is, an antigen-antibodycomplex, and an amount of antigen-antibody complex formation may bequantitatively measured from the size of a signal of a detection label.The detection level may be selected from the group consisting of anenzyme, a fluorescent material, a ligand, a light-emitting material, amicroparticle, a redox molecule and a radioisotope, but the presentinvention is not limited thereto. Analysis methods for measuring aprotein level may include, but not limited to, Western blotting, ELISA,radioimmunoassay, radioimmunodiffusion, Ouchterlonyimmunodiffusion,rocket immunoelectrophoresis, immunohistochemical staining,immunoprecipitation assay, complement fixation assay, FACS, and proteinchip assay.

Accordingly, the present invention may confirm mRNA expression levels orprotein amounts in the control, a patient resistant to an EGFR-targetingagent, or a patient suspected of having resistance to an EGFR-targetingagent through the detection methods described above, and the expressionlevels are compared to be able to diagnose the occurrence andprogression of the resistance to an EGFR-targeting agent.

In addition, according to the method of providing information for thediagnosis of the resistance to an EGFR-targeting agent according to thepresent invention, when the expression level of the CD133 gene accordingto the present invention or an amount of the expressed protein is higherthan that of the normal control sample, it can be determined that apatient has resistance to an EGFR-targeting agent.

The present invention also relates to a composition for preventing ortreating resistance to an EGFR-targeting agent, which includes a CD133inhibitor.

The present invention also provides a use of a CD133 inhibitor forpreparing a pharmaceutical composition for preventing or treatingresistance to an EGFR-targeting agent.

According to the present invention, CD133 is highly expressed in cancercells with resistance to an EGFR-targeting agent, as described above,and the expression of CD133 promotes budding of microvesicles, increasescell migration and invasiveness by delivering the oncogenic protein KRASG13D to adjacent cells using microvesicles, and thus is involved in theoccurrence of anticancer agent resistance.

Therefore, the CD133 inhibitor may be used in treatment of resistantcancer.

To this end, the composition for preventing or treating resistance to anEGFR-targeting agent of the present invention may include an agent forreducing mRNA expression of the CD133 gene or protein expressionthereof, or degrading function or activity.

The CD133 protein inhibitor may be a peptide or compound which is boundwith the CD133 protein to regulate a signal of a nerve differentiationpathway. The inhibitor may be selected by a screening method exemplifiedbelow, such as protein structure analysis, and may be designed using amethod known in the art.

Specifically, the CD133 protein inhibitor may be a material which isbound with the CD133 protein consisting of an amino acid sequencerepresented by SEQ ID NO: 2 to inhibit activity.

In addition, as the protein inhibitor, a polyclonal antibody, monoclonalantibody, human antibody or humanized antibody against the CD133 proteinmay be used, and the definition of the antibody is as described above.

Resistant cancer may be prevented or treated by inhibiting the functionof CD133 in cells using the antibody.

The functional or activity inhibitor of the CD133 protein according tothe present invention may be delivered using a liposome, a virus, a genegun, a polymer, ultrasonication or an electric shock, but the presentinvention is not particularly limited thereto.

The CD133 gene may be DNA encoding the same or mRNA transcribedtherefrom. Accordingly, the inhibitor of the gene may be an inhibitorwhich is bound to the gene itself to interfere with transcription orbound to mRNA transcribed from the gene to interfere with thetranslation of mRNA.

Therefore, the inhibitor of the CD133 gene includes all types ofinhibitors that inhibit the expression of the CD133 gene. For example,such an inhibitor may be a peptide, nucleic acid or compound binding tothe gene. The inhibitor may be selected by a screening method to bedescribed below, such as cell-based screening, and may be designed by amethod known in the art.

In one embodiment, the inhibitor may be an antisense-oligonucleotide,siRNA, shRNA or miRNA against the CD133 gene or a vector including thesame. Such an antisense-oligonucleotide, siRNA, shRNA, miRNA or a vectorincluding the same may be manufactured using a method known in the art.Specifically, the inhibitor may be a nucleic acid molecule prepared toinhibit the expression of the CD133 gene consisting of SEQ ID NO: 1.

The term “siRNA” used herein refers to double-stranded RNA inducing RNAinterference through the cleavage of mRNA of a target gene, and consistsof an RNA strand of a sense sequence having the sequence like mRNA ofthe target gene and an RNA strand of an antisense sequence having asequence complementary thereto.

The siRNA may include siRNA synthesized in vitro, or a form expressed byinserting a base sequence encoding siRNA into an expression vector.

The “vector” used herein refers to a gene construct including foreignDNA inserted into a genome encoding a polypeptide.

The vector related to the present invention is a vector in which anucleic acid sequence inhibiting the gene is inserted into a genome, andsuch a vector may be a DNA vector, a plasmid vector, a cosmid vector, abacteriophage vector, an enzyme vector, or a viral vector.

In addition, the antisense may have a sequence complementary to anentire or partial mRNA sequence transcribed from the CD133 gene or afragment thereof, and bind to the mRNA to inhibit the expression of theCD133 gene or a fragment thereof.

In addition, the short hairpin RNAi (shRNAi) may be manufactured by aconventional method using a human or mouse shRNAi common base sequenceregion as a target.

In addition, the pharmaceutical composition of the present invention mayfurther include a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier includes a carrier and avehicle, which are generally used in the pharmaceutical field, andspecifically, includes an ion exchange resin, alumina, aluminumstearate, lecithin, a serum protein (e.g., human serum albumin), abuffer material (e.g., various types of phosphates, glycine, sorbicacid, potassium sorbate, or a partial glyceride compound of a saturatedvegetable fatty acid), water, a salt or electrolyte (e.g., protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride or a zinc salt), colloidal silica, magnesiumtrisilicate, polyvinylpyrrolidone, a cellulose-based substrate,polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax,polyethylene glycol or lanolin, but the present invention is not limitedthereto.

In addition, the composition of the present invention may furtherinclude a lubricant, a wetting agent, an emulsifier, a suspending agent,or a preservative, other than the above-described components.

In one aspect, the composition according to the present invention may beprepared as an aqueous solution for parenteral administration, andpreferably, a Hank's solution, a Ringer's solution or a buffer solutionsuch as physically buffered saline is used. An aqueous injectablesuspension may include a substrate that can increase the viscosity ofthe suspension such as sodium carboxymethylcellulose, sorbitol ordextran.

The composition of the present invention may be systemically or locallyadministered, and formulated in a suitable form by known technology forsuch administration. For example, for oral administration, thecomposition may be administered by mixing the active compound with aninactive diluent or edible carrier, encapsulating all components with ahard or soft gelatin capsule or compressing all components in the formof a tablet. For oral administration, the active compound may be mixedwith an excipient, and then formed as an edible tablet, a buccal tablet,a troche, a capsule, an elixir, a suspension, a syrup or a wafer.

Various forms for injection or parenteral administration may be preparedaccording to a technique known or commonly used in the art. Since CD133is well dissolved in saline or a buffer solution, after storage in afreeze-dried state, an effective amount of CD133 may be mixed in salineor a buffer solution to prepare a solution suitable for intravenousinjection, subcutaneous injection, muscle injection, intraperitonealinjection or transdermal injection, right before the administration.

The effective amount of the active ingredient of the pharmaceuticalcomposition of the present invention refers to an amount required forprevention, inhibition or alleviation of a disease.

Accordingly, the effective amount may be adjusted by various factorssuch as the type and severity of a disease, the type and content of theactive ingredient and other components, contained in the composition,the type of a dosage form, and the age, body weight, general healthcondition, gender and diet of a patient, administration time,administration route, a release rate of the composition, a treatmentperiod, and a co-used drug. While not limited thereto, for example, inthe case of an adult, the inhibitor of the present invention may beadministered once or several times a day, when the type of the inhibitoris a compound, it may be administered at a dose of 0.1 ng/kg to 10 g/kg,when the type of the inhibitor is a polypeptide, protein or antibody, itmay be administered at a dose of 0.1 ng/kg to 10 g/kg, or when the typeof the inhibitor is an antisense-oligonucleotide, siRNA, shRNAi ormiRNA, it may be administered at a dose of 0.01 ng/kg to 10 g/kg.

The present invention also provides a method of treating an animal withresistance to an EGFR-targeting agent, which includes administering acomposition including a pharmaceutically effective amount of a CD133inhibitor for preventing or treating resistance to an EGFR-targetingagent to a subject.

A pharmaceutical composition and an administration method, which areused in the method of treating resistance to an EGFR-targeting agent,are as described above, and the common description between them will beomitted to avoid excessive complexity of the specification.

Meanwhile, subjects to which the pharmaceutical composition forpreventing or treating resistance to an EGFR-targeting agent may beadministered include all kinds of animals. For example, the subjects maybe non-human animals such as dogs, cats, and mice.

The present invention also provides a method of screening a drug forpreventing or treating resistance to an EGFR-targeting agent, whichincludes bringing the CD133 gene into contact with a candidate ex vivo,and determining whether the candidate promotes or inhibits theexpression of the gene.

In addition, the present invention provides a method of screening a drugfor preventing or treating resistance to an EGFR-targeting agent, whichincludes bringing the CD133 protein into contact with a candidate exvivo, and determining whether the candidate improves or inhibits thefunction or activity of the protein.

According to the screening method of the present invention, first, acandidate to be analyzed may be brought into contact with resistantcancer cells having the gene or protein.

The candidate may be an individual nucleic acid, protein or peptide,another extract or natural substance, or a compound, which is expectedor randomly selected to have a potential as a material promoting orinhibiting the mRNA transcription or mRNA translation into a protein inthe base sequence of the CD133 gene or a drug improving or inhibitingthe function or activity of the CD133 protein according to a commonselection method.

Afterward, in cells treated with a candidate, the expression level ofthe gene or the amount or activity of the protein may be measured, andas a result of the measurement, when the expression level of the gene orthe amount or activity of the protein is measured as increased ordecreased, the candidate may be determined as a material for treating orpreventing resistant cancer.

The method of measuring the expression level of the gene, or the amountor activity of the protein may be performed by various methods known inthe art, and may be, for example, RT-PCR, real time-polymerase chainreaction, Western blotting, Northern blotting, ELISA, radioimmunoassay(RIA), radioimmunodiffusion and immunoprecipitation assay, but thepresent invention is not limited thereto.

The candidate exhibiting an activity of inhibiting gene expression or aprotein function, obtained by the screening method of the presentinvention, may be a candidate for a resistant cancer therapeutic agent.

The candidate for a therapeutic agent against cancer resistant to anEGFR-targeting agent serves as a leading compound in the development ofa therapeutic agent against resistance to an EGFR-targeting agent, andmodifies and optimizes the structure of the leading compound to exhibitan effect of inhibiting the CD133 gene or the function of a proteinexpressed therefrom, thereby developing a novel therapeutic agentagainst cancer resistant to an EGFR-targeting agent.

Details related to genetic engineering technology in the presentinvention will become more apparent from the content disclosed inSambrook, et al. Molecular Cloning, A Laboratory Manual, Cold SpringHarbor laboratory Press, Cold Spring Harbor, N.Y. (2001); and FrederickM. Ausubel et al., Current protocols in molecular biology volume 1, 2,3, John Wiley & Sons, Inc. (1994).

ADVANTAGEOUS EFFECTS

The present invention identifies that the CD133 protein expressed incolon cancer increases resistance to an EGFR-targeting agent, and thusthe CD133 protein can be used as a novel target protein for diagnosingand treating resistant cancer as well as general cancer.

DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1E show the result of identifying a CD133 expression regulatorymechanism in liver cancer cells: (A) the change in CD133 expressionaccording to EGF expression; (B-C) the change in CD133 expressionaccording to an EGF downstream signaling molecule, NF-κB; and (D-E) therelationship between NF-κB and CD133, confirmed by promoter activity.

FIGS. 2A-2E show the result of confirming that CD133 is involved inpromotion of microvesicle budding in liver cancer cells: (A)confirmation of the relationship between an EGF signaling system andmicrovesicle and TNT formation; (B) confirmation of microvesicle and TNTformation in a CD133—overexpressing cell line; and (C-E) confirmation ofthe change in activation of Rac1 and RhoA associated with microvesiclebudding and a downstream signaling factor, Erk1/2, of RhoA according toCD133 expression

FIGS. 3A-3B show the result of analyzing microvesicles budded fromcancer cells: (A) the result of confirming whether CD133 is included inmicrovesicles budded from various cancer cells; and (B) the result ofobserving the migration of microvesicles containing CD133 to adjacentcells.

FIGS. 4A-4F show the result of confirming that CD133 is involved inoncogenic protein transport and microvesicle budding in colon cancer:(A) confirmation whether an oncogenic protein is contained inmicrovesicles according to CD133 expression; (B-C) confirmation thatCD133 expression contributes to microvesicle budding and a size change;(D) confirmation that CD133 expression contributes to the transport ofan oncogenic protein via microvesicles and cell proliferation; and (E-F)confirmation that the transport of an oncogenic protein viamicrovesicles contributes to the change in cell migration to andinvasiveness of adjacent normal cells.

FIGS. 5A-5F show the result of confirming that CD133—containingmicrovesicles induce anticancer agent resistance (gefitinib) in coloncancer: (A-B) confirmation of the capability of CD133—containingmicrovesicles to induce anticancer agent resistance (gefitinib) byconfirmation of the proliferation of cancer cells; (C-D) the result ofconfirming that cancer cell proliferation is caused by an oncogenicprotein transported by microvesicles through the change in KRASdownstream signaling molecule and target gene expression; and (E-F)confirmation that cell migration to and invasiveness of adjacent normalcells are changed by the oncogenic protein transported byCD133—containing microvesicles in the presence of gefitinib.

MODES OF THE INVENTION

Hereinafter, the advantages and features of the present invention andthe methods of accomplishing the same may be clearly understood withreference to the detailed description of exemplary embodiments and theaccompanying drawings. However, the present invention is not limited tothe exemplary embodiments disclosed below, and may be embodied in manydifferent forms. These exemplary embodiments are merely provided tocomplete the disclosure of the present invention and fully convey thescope of the present invention to those of ordinary skill in the art,and the present invention should be defined by only the accompanyingclaims.

<Example 1>Confirmation of CD133 Expression Regulatory Mechanism inCancer Cells

An epithelial growth factor (EGF) signaling system has been reported tobe significant in the occurrence and development of cancer. CD133 (NCBIGene ID: 8842) is known as a marker of a cancer stem cell, andparticularly, to play a crucial role in formation of liver cancer stemcells. Therefore, the correlation between the EGF signaling system andCD133 expression in a liver cancer cell line was investigated.

After treatment with an inhibitor against an EGF downstream signalingmolecule, CD133 expression was investigated. Specifically, fortransfection and Western blotting, a specific gene (plasmid vector orsiRNA) was expressed in cells using a transfection reagent. For generalplasmid vector transfection, 24 hours after transfection, cells wereharvested, and for siRNA transfection, 48 hours after transfection,cells were harvested. For Western blotting, the harvested cells werelysed in RIPA buffer, and centrifuged at 12,000 g for 20 minutes at 4°C., followed by collecting a supernatant. The collected supernatant wassubjected to SDS-PAGE gel electrophoresis, and transferred onto anitrocellulose membrane, followed by detecting the expression of adesired protein using suitable antibodies. As a result, it was observedthat NF-κB is involved in EGF-induced CD133 expression (FIG. 1B). It wasconfirmed that, when the expression of NF-κB subunits, such as p50 (NCBIGene ID: 4790) and p65 (NCBI Gene ID: 5970), was inhibited, CD133expression decreases (FIG. 1C). Promoter activity was observed in apromoter near CD133 ORF. Specifically, to measure the promoter activity,a CD133 ORF promoter region was cloned in a special vector expressingluciferase. Cells were simultaneously transfected with the luciferasevector and a Renilla plasmid vector for quantification. Twenty-fourhours after transfection, the cells were treated with EGF for 14 hours,lysed with lysis buffer and centrifuged to obtain a supernatant, andthen luciferase activity was measured using the Luminometer 20/20 (FIG.1D). In a promoter which had been subjected to treatment with an NF-κBinhibitor and an NF-κB subunit-binding site, promoter activity was notobserved (FIG. 1D). Taken together, it was confirmed that EGF activatesNF-κB and regulates CD133 expression at the transcription level (FIG.1A).

<Example 2>Identification of CD133 Role in Cancer Cells

The relationship between EGF and microvesicle and TNT formation wasinvestigated by microscopic analysis. Specifically, after 14 hours ofEGF treatment, cells were fixed with 4% paraformaldeyde and treated withWGA-488 and DAPI for 10 minutes to stain the cell membrane and nucleus.Fluorescence intensity was measured using an LSM700 Meta confocalmicroscope.

The results showed that, when a liver cancer cell line was treated withEGF, compared to a control, microvesicle and TNT formation increasesbetween cells, and this phenomenon is decreased by treatment with theNF-κB inhibitor (WGA, cell membrane staining, FIG. 2A). In addition, ina CD133 expression-stable cell line, it was confirmed that microvesiclesand TNT formation rates highly increase (FIG. 2B). Therefore, it wasexpected that microvesicle budding is closely related to the change inCD133 expression.

In addition, to bud microvesicles from the cell membrane, the regulationof activities of small GTPases such as ARF6, RhoA and Rac1 is known tobe important, and thus an expression pattern of the gene according tothe CD133 expression pattern was observed by small GTPase pull-downassay. Specifically, to measure Rac1 activity, after transfected withCD133, cells were harvested, and activity was measured using a Rac1activation Assay kit. To measure RhoA activity, after CD133transfection, cells were harvested, and activity was measured using aRhoA activation Assay kit. As a result, it was seen that, in the CD133expression-stable cell line, Rac1 (NCBI Gene ID: 5879) activitydecreases, RhoA (NCBI Gene ID: 387) activity increases, and thus theactivity of the downstream signaling molecule Erk1/2 (NCBI Gene ID:5595, 5594) increases (FIG. 2E). This result showed that a certain levelof CD133 expression is essential for microvesicle formation, andparticularly, small GTPase activity is regulated to promote microvesiclebudding.

<Example 3>Analysis of Physiological Properties of MicrovesiclesReleased from Cancer Cells

Microvesicles were observed by microvesicle microscopic analysis.Specifically, microvesicles were isolated from the culture solution ofCD133-transfected cells and stained with WGA-488, general cells weretreated with the stained microvesicles, and after 12 hours, fluorescenceintensity was measured using a LSM700 Meta confocal microscope.

It was observed that the microvesicles were released from various typesof cancer cells, and contained CD133 (FIG. 3A). In addition, the CD133(red)-containing microvesicles were isolated by centrifugation, andtreated with WGA to stain the cell membrane. It was observed that, whenthe cell line was treated with the microvesicles, the microvesicles weretransported to adjacent cells along with CD133 (FIG. 3B).

<Example 4>Identification of CD133 Role in Colon Cancer

Forty-eight hours after a HCT116 cell line was transfected with ashCD133 vector into which the shRNA sequence (5′:GAGUCGGAAACUGGCAGAUAGCAAU-3′: SEQ ID NO: 3) knocking down the CD133expression was inserted to prepare a CD133 expression-inhibited cellline, selection was performed with a selection marker Zeocin. Afterward,single colonies were selected and subcultured, and subjected to Westernblotting with wild-type HCT116 to verify CD133 knockdown. The usedvector is a vector prepared by substituting a neomycin antibiotic regionwith Zeomycin in a pSilencer 2.1-U6 neo vector (www.thermofisher.com/kr/ko/home/life-science/dna-rnapurification-analysis/napamisc/vector-maps/psilencer-2-1-u6-hygro-vectormap.html).

To isolate microvesicles, the culture solutions of a CD133 normalexpression cell line and a CD133 expression-inhibited cell line werecollected, and then each cell culture solution was centrifuged at 20,000g for 1 hour, thereby obtaining a supernatant. After discarding thesupernatant, microvesicles were isolated.

From the isolated microvesicles, the amount and sizes of microvesiclebudding were measured by a Malvern Nanosight Nanoparticle TrackingAnalysis system.

KRAS (NCBI Gene ID: 3845) G13D is a well-known oncogenic protein, andusually found as a KRAS mutant in colon cancer. It was observed that,when CD133 expression was inhibited in a colon cancer cell line, KRASG13D was not contained in the microvesicles (FIG. 4A). In addition, itwas confirmed that CD133 expression is involved in determination of theamount and sizes (100 to 200 nm) of microvesicle budding (FIGS. 4B and4C).

To observe the transport of KRAS G13D to adjacent cells, microvesicleswere isolated from the CD133 expression-inhibited cell line and thegeneral cell line and then normal cells were treated with themicrovesicles. Specifically, for cell migration assay,microvesicle-recipient cells were cultured in an 8-μm pore insert,treated with microvesicles, and then after 48 hours, migrating cellscounted. For invasion assay, an 8-μm pore insert was coated withMatrigel, and then recipient cells were incubated. Forty-eight hoursafter microvesicle treatment, cells migrating through the Matrigel werecounted.

As a result, it was confirmed that, in normal cells treated with themicrovesicles isolated from the general cell line, KRAS G13D istransported to adjacent cells via the microvesicles along with CD133,and the activation of KRAS downstream signaling molecules Akt (NCBI GeneID: 207) and Erk1/2 increases (FIG. 4D). The microvesicle-mediated KRASG13D transport induced increases in cell migration to (FIG. 4E) andinvasiveness (FIG. 4F) of normal cells. Based on this result, it wasable to be confirmed that CD133 regulates a microvesicle transportmaterial, and plays a crucial role in microvesicle size and budding.

<Example 5>Confirmation of Induction of Anticancer Agent Resistance toAdjacent Cells by CD133—Containing Microvesicles in Colon Cancer

While gefitinib is an anticancer material that inhibits EGFR activityand thus controls cancer cells, it was reported that, in certain typesof cancer in which EGFR downstream signaling molecules including KRASare activated, gefitinib efficacy was insignificant. It was inferredthat the transport of KRAS G13D to adjacent cells by CD133-containingmicrovesicles is involved in resistance to such an EGFR-targeting agent.

Cells being cultured to confirm the above inference were incubated withgefitinib and the microvesicles for a suggested time, cell viability wasanalyzed using an EZ-cytox kit at specific time, and the absorbance wasmeasured at 570 nm using a microplate reader, followed by calculating acell growth rate. Twenty-four hours after treatment with themicrovesicles and gefitinib, the cells were harvested, and then fixedwith 95% cold ethanol. The fixed cells were treated with RNase andpropidium iodide to stain DNA, and then the stained DNA was analyzed byFACS.

As a result, it was confirmed that, when treated along with gefitinib,microvesicles extracted from CD133 expression-inhibited colon cancercells do not contribute to cell proliferation of adjacent recipientcells, but microvesicles extracted from CD133-expressing cells restorecell proliferation even when gefitinib is treated (FIGS. 5A and 5B). Todemonstrate that such a change is caused by KRAS G13D transport bymicrovesicles, the change in KRAS downstream signaling molecules andtarget genes in microvesicle-recipient cells was observed. It wasconfirmed that the CD133-mediated KRAS G13D transport increases theactivities of KRAS downstream signaling molecules FAK (NCBI Gene ID:5747), Akt and Erk1/2 in the recipient cells (FIG. 5C). In addition, itwas observed that mRNA expressions of KRAS target genes SERVIVIN (NCBIGene ID: 332) and CCNB1 (NCBI Gene ID: 891) increase, anti-apoptoticmRNA (BCLXL, BCL2L2 (NCBI Gene ID: 598, 599)) increases, andpro-apoptotic mRNA (BIM (NCBI Gene ID: 10018)) decreases (FIG. 5D). Thisshows that the cell migration to and invasiveness of recipient cellsreceiving the oncogenic protein (KRAS G13D)—containing microvesiclesincrease, and thus the development of cancer is maintained aftertreatment with an anticancer agent. In conclusion, in colon cancer,CD133—containing microvesicles are involved in transport of the KRASoncogenic protein to induce anticancer agent resistance to adjacentcells, thereby accelerating cancer development.

1. A method of treating resistance to Gefitinib in colon cancer, themethod comprising administering an inhibitor against a CD133 gene to asubject resistant to Gefitinib, wherein the inhibitor is a peptide, acompound, or an antibody against the CD133 protein.